Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases

ABSTRACT

Compounds and methods for designing and identifying compounds which inhibit TFPP-like aspartyl protease enzymes by targeting the aspartic acid residues of the active site or mimicking peptides corresponding to the region surrounding the substrate&#39;s cleavage site are provided. Agents identified as inhibitors of TFPP-like aspartyl proteases such as type 4 prepilin peptidases are expected to be useful as anti-bacterial agents and in inhibiting development of drug resistant strains of bacteria.

This application claims the benefit of provisional application Ser. No.60/143,355 filed Jul. 12, 1999.

This invention was supported in part by funds from the U.S. government(NIH Grant No. RO1 AI25096 and F31 AI09635) and the U.S. government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

A family of transmembrane protease enzymes has now been identified whichcomprise two critical aspartic acid residues on the same side of themembrane at their active cleavage site. Members of this family ofprotease inhibitors can be identified by a consensus sequence at theC-terminal aspartic acid residue comprising G-X-G-D-(F or V or P or K orI or L or Y). The mechanism of action of these aspartyl proteases isexemplified herein through the bacterial protease type 4 prepilinpeptidase (TFPP). Accordingly, these proteases are referred to herein asTFPP-like aspartyl proteases. The present invention relates tocompositions and methods for identifying and designing compositionswhich inhibit TFPP-like aspartyl proteases by targeting the asparticacid residues of the enzymatic active site or mimicking a regionsurrounding the cleavage site of a TFPP-like aspartyl proteasesubstrate. These compositions are expected to be useful in inhibitingthe activity of TFPP-like aspartyl proteases involved in diseases andinfections.

BACKGROUND OF THE INVENTION

The development of the first anti-infective or anti-bacterial agents inthe early 1900's and the success seen with their use led to thedevelopment of hundreds of new compounds to fight the wide variety ofbacterial organisms. Most people have had exposure to these drugs atsome point in their life, with the majority of situations resulting in arapid cure of the bacterial infection with relatively few side effects.However, human morbidity and mortality due to bacterial infections hasbecome a major concern today due to the dramatic increase in thefrequency of infections caused by bacteria that are resistant to most,if not all, of the available anti-bacterial agents. This increase indrug-resistant pathogens has led to an ever-increasing need for newdrugs with new mechanisms of action.

Anti-bacterial agents are developed by identifying unique targets notpresent in mammalian cells and then designing a drug to exploit thatdifference such that the bacterial cells are killed or neutralized whilemammalian cells are left intact and unaffected. The goal of successfulanti-bacterial drug therapy is to limit toxicity in the patient whilemaximizing the ability of the drug to invade the bacterial cells andneutralize those cells as selectively as possible. The major classes ofanti-bacterial drugs available today target a variety of differentcellular components and functions of bacteria such as the cell wall,protein synthesis, cell metabolism, DNA synthesis, and the bacterialcell membrane. Each of these target cellular components or functions isrelated in some way to the disease process of bacterial infections thatinvolves first colonization of the bacteria, invasion of host cells,production of cellular toxins or inflammatory agents, and a hostresponse to those toxins or agents.

A fundamental process of all living cells, including bacteria, is thesecretion of proteins across membranes. The majority of proteins thatare secreted are synthesized as a precursor with an N-terminal signalsequence (or leader peptide) of about 16-24 amino acids in length. Thisleader sequence serves to promote recognition of the protein by thesecretory apparatus of the cell and facilitates movement across themembrane. The leader sequence is subsequently processed by a leaderpeptidase to remove the sequence and allow release of the mature oractive protein. Recent research has indicated that in the case ofbacteria, there are several systems for secreting proteins and some ofthese systems have unique leader peptidases associated with theircognate secreted proteins. One of these systems is known as the type 2secretion system which promotes extracellular secretion of bacterialfactors such as toxins and colonization pili that are the hallmarks ofthe mechanisms that promote virulence of pathogenic bacteria. Pilimediate the binding of bacteria to host tissues and most pili arecomposed of a major protein subunit that polymerizes to form a pilus.

The type 2 secretion systems of most bacteria involve a type 4 pilin forcolonization pilus formation and type 4 pilin-like proteins forsecretion of toxins and other factors associated with bacterialvirulence and destruction of host tissue and enhancement of bacterialgrowth in the host. Highly related type 4 pili serve as the majorcolonization factors for up to 50 different gram-negative bacterialspecies and type 4 pilin-like proteins have been found for a growingnumber of gram-positive bacteria as well. Type 4 pili are composed of apolymerized structure of type 4 pilin. The pilin is synthesized as aprepilin with a leader peptide that is very different from those oftypical secreted proteins. A type 4 specific leader peptidase isrequired to process a type 4 prepilin leader sequence to allow secretionof the mature protein. Importantly, this secretion system including thetype 4 leader peptidase itself is only found in bacteria and is notpresent in humans or other potential hosts of infection. Furthermore,mutating the type 4 prepilin peptidase (TFPP) renders the bacteriumavirulent (March and Taylor 1998. Mol. Microbiol. 29:1481-1492).

The type 4 signal peptide is highly conserved across all type 4 prepilinor prepilin-like proteins and is composed of 6 to 25 highly chargedamino acids at the N-terminus followed by approximately 20 predominatelyhydrophobic amino acids. Cleavage occurs between the two domainsimmediately C-terminal of an invariant glycine and before the newN-terminal amino acid that is usually a methionine or a phenylalanine.Unlike cleavage of standard signal peptide by signal peptidase I,wherein the cleavage occurs on the periplasmic side of the innermembrane, processing by a type 4 peptidase occurs on the cytoplasmicside of the inner membrane (Strom and Lory, 1993. Ann. Rev. Microbiol.47:565-596). Previous mutational analysis and protease inhibitorevidence from studies of pilD of Pseudomonas aeruginosa and proteinalignment analysis of the type 4 peptidase family suggested two pairs ofcysteines in cytoplasmic domain 1, the largest cytoplasmic domain, to beinvolved in the protease active site of the enzyme (Strom et al. 1993.Proc. Natl. Acad. Sci. USA 90:2404-2408). These data resulted in thecategorization of type 4 prepilin peptidase (TFPP) family as a type ofcysteine protease (Strom et al. 1994. Meth. Enzymol. 235:527-540).

However, subsequent studies have shown the two cysteine pairs to belacking in XpsO, a type 4 prepilin peptidase of X. campestris. In fact,several type 4 prepilin peptidases, among the approximately 27 that havebeen identified and cloned, do not have the conserved cysteines in theirprotein sequence but still function to cleave the type 4 signal peptide.

In the present invention a specific domain of type 4 prepilin peptidasethat is essential for cleavage activity has now been identified to theresolution of two specific amino acids. Identification of this domainhas facilitated the identification of specific inhibitors of theprotease activity of type 4 prepilin peptidase as well as otherTFPP-like aspartyl proteases which utilize this same cleavage mechanism.

SUMMARY OF THE INVENTION

Using mutant constructs of TcpJ, residues essential for cleavageactivity of the bacterial protease type 4 prepilin peptidase (TFPP) havenow been identified as two aspartic acid residues. Thus, usefulanti-bacterial agents targeted to these residues can now be designed toinhibit the cleavage activity of type 4 prepilin peptidase which isessential to growth of bacteria in a host. Further, a consensus sequenceat the C-terminal aspartic acid residue comprising G-X-G-D-(F or V or Por K or I or L or Y) has been identified so that TFPP-like aspartylproteases of organisms other than bacteria, including humans, whichcomprise this novel active site and utilize a cleavage mechanism similarto that exemplified herein for type 4 prepilin peptidase can beidentified. For purposes of the present invention, proteases of this newfamily which are transmembrane protease enzymes comprising two criticalaspartic acid residues on the same side of the membrane at their activecleavage site, wherein a consensus sequence at the C-terminal asparticacid residue comprising G-X-G-D-(F or V or P or K or I or L or Y) ispresent, are referred to herein as “TFPP-like aspartyl proteases”. Thisterm is meant to be inclusive of type 4 prepilin peptidases.

Accordingly, an object of the present invention is to provide a methodof designing inhibitors of TFPP-like aspartyl proteases which comprisessynthesizing compounds which target the aspartic acid residues of theenzymatic active site of the TFPP-like aspartyl protease or mimic aregion surrounding the cleavage site of the TFPP-like aspartyl proteasesubstrate and inhibit cleavage activity of the protease. Also providedin the present invention are methods of selecting and screeningcompounds for inhibitory activity of these TFPP-like aspartyl proteaseenzymes. Compounds identified as inhibitors can be used to modulateactivity of TFPP-like aspartyl proteases involved in disease andinfection. In one embodiment, compounds identified as inhibitors areused as anti-bacterial agents in the inhibition of type 4 prepilinpeptidases.

Thus, another object of the present invention is to provide newanti-bacterial agents which comprise compounds which target the asparticacid residues of type 4 prepilin peptidase and inhibit cleavage activityof this peptidase. As will be obvious to those of skill in the art uponthis disclosure, such agents are also expected to be useful ininhibiting other TFPP-like aspartyl protease enzymes of this familywhich utilize the same cleavage mechanism as described herein for type 4prepilin peptidase and/or contain the same active site. Other TFPP-likeaspartyl protease enzymes of this family can be identified routinely bythose of skill in the art based upon the presence of the consensussequence comprising G-X-G-D-(F or V or P or K or I or L or Y) at theC-terminal aspartic acid residue of the enzymatic active site.

Another object of the present invention is to provide methods ofinhibiting activity of TFPP-like aspartyl protease enzymes viaadministration of a compound which targets the aspartic acid residues ofthe enzymatic active site of the TFPP-like aspartyl protease or mimics aregion surrounding the cleavage site of the TFPP-like aspartyl proteasesubstrate and inhibits cleavage activity of the protease. In oneembodiment of the present invention, bacterial infection in a host isinhibited by administering to a host infected with the bacteria acompound which targets the aspartic acid residues of type 4 prepilinpeptidase and inhibits cleavage activity of this peptidase.

Also related to this embodiment of the present invention are methods andcompositions for decreasing development of drug resistant strains ofbacteria through coadministration of a compound which targets theaspartic acid residues of type 4 prepilin peptidase and inhibitscleavage activity of this peptidase with a second known therapeuticallyeffective anti-bacterial agent.

Another object of the present invention is to provide TcpJ mutantconstructs useful in defining the active cleavage site of type 4prepilin peptidase and in X-ray crystal structure analysis of thisenzyme.

Yet another object of the present invention is to provide a homologoustype 4 prepilin peptidase gene identified in Staphylococcus aureus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bargraph showing the percent of conserved homology ofpotential protease active site residues from 27 type 4 prepilinpeptidase homologs. Conserved residues which do not exist in TcpJ are inparenthesis and are designated with the relative position in TcpJ.Predicted membrane topology is indicated by C (cytoplasmic), P(periplasmic) or M (transmembrane) below each residue. Shaded barsindicate the conserved residues which were altered in experimentsdescribed herein. Alignment was performed in DNASTAR MegAlign using theClustal method.

FIG. 2 is a predicted TcpJ membrane topology model. The predictedtopology of TcpJ was deduced by comparison with OutO of Erwiniacarotovora for which the membrane topology has been determined by theuse of beta lactamase-fusions in outO. Cytoplasmic domains 1, 2 and 3have been designated as well as C (cytoplasmic), P (periplasmic) and M(transmembrane) regions. All potential protease active sites (C, S, D,K, H, E) have been designated. Amino acids altered in experimentsdescribed herein are shaded and numbered by their position. Eachcritical active site residue, D125 and D189, is marked with a “*”.

DETAILED DESCRIPTION OF THE INVENTION

The type 4 prepilin peptidase is found only in bacteria and isresponsible for the cleavage and N-methylation of a large number ofsecreted proteins known as type 4 prepilins, or prepilin-like proteins,that have a type 4 signal peptide at their N-terminus. The cleavage ofthe type 4 signal peptide is a necessary step to secretion of the type 4prepilin. Prepilin-like proteins are involved in DNA competence and/oruptake in B. subtilis, in phage morphogenesis in E. coli, and partiallycomprise the type 2 secretion system which is the main terminal branchof the general secretory pathway in gram negative bacteria (Pugsley,1993. Microbiology Reviews 57(1):50-108). Type 4 pili serve as the majorcolonization factors for up to 50 different gram-negative bacterialspecies. Therefore, agents which interfere with, or inhibit, activity oftype 4 prepilin peptidase are expected to be useful as anti-bacterialdrugs and in the identification of anti-bacterial drugs. Further, giventhe role of prepilin-like proteins in DNA uptake, particularly ingram-positive bacteria (Dubnau, D. 1997. Gene 192:191-198; Wolfgang etal. 1999. Molecular Microbiology 31(5):1345-1357), it is believed thatcoadministration of these agents with a second anti-bacterial agent willinhibit uptake of antimicrobial agent resistance gene thereby reducingdevelopment of drug resistant strains of bacteria.

It has now been found that type 4 prepilin peptidase, specifically TcpJ,a type 4 prepilin peptidase of Vibrio cholerae, is a non-pepsin-likeacid protease. A homologous gene has also now been identified inStaphylococcus aureus, a bacterium which is very difficult to treat dueto its ability to become resistant to drugs. The nucleic acid sequenceencoding this peptidase in Staphylococcus aureus is depicted as SEQ IDNO:1. The amino acid sequence of this Staphylococcus aureus peptidase isdepicted as SEQ ID NO:2. Contrary to prior art suggestions of cysteineresidues being involved in the protease active site of this enzyme((Strom et al. 1993. Proc. Natl. Acad. Sci. USA 90:2404-2408), residuesessential for cleavage activity of this peptidase have now beenidentified as two aspartic acid residues. Further, a consensus sequenceat the C-terminal aspartic acid residue comprising G-X-G-D- (F or v or Por K or I or L or Y) has been identified so that TFPP-like aspartylproteases of organisms other than bacteria, including humans, whichcomprise this novel active site and utilize a cleavage mechanism similarto that exemplified herein for type 4 prepilin peptidase can beidentified. Accordingly, this peptidase is part of a novel subclass orfamily of non-pepsin-like acid proteases, referred to herein asTFPP-like aspartyl proteases. For purposes of the present invention, by“TFPP-like aspartyl protease” it is meant a transmembrane proteaseenzyme comprising two critical aspartic acid residues on the same sideof the membrane at its active cleavage site, wherein a consensussequence at the C-terminal aspartic acid residue comprising G-X-G-D-(For V or P or K or I or L or Y) is present. This term is meant to beinclusive of type 4 prepilin peptidases.

Further, it has now been demonstrated that agents which target theaspartic acid residues of this active site inhibit the cleavage activityof proteases in this new family. For example, inhibition of type 4prepilin peptidase was accomplished by contact of the peptidase with thecombination of EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride) and glycinamide, a combination of compounds known toinhibit non-pepsin-like acid proteases. Using the knowledge of thistarget area within the peptidase sequence essential for cleavageactivity, additional inhibitors of this class of protease enzymes canalso be identified. For example, inhibitors designed to target theaspartic acid residues, D125 and D189, which are essential to thecleavage activity of type 4 prepilin peptidase, can be synthesized forse as anti-bacterial agents. Preferably, the inhibitors are transitionstate analogues or other compounds that modify or competitively inhibitthe aspartic acid residues thereby preventing cleavage activity.

To determine the particular amino acids of aspartyl proteases such asTcpJ and other type 4 prepilin peptidases that act as the catalyticresidues of the protease activity, the protein sequence alignment of 27type 4 prepilin peptidases was examined for highly conserved amino acidswhich have the potential to be protease active site residues. Allcysteines, serines, aspartic acids, histidines, lysines, and glutamicacids that represent consensus residues in these type 4 prepilinpeptidases are shown in FIG. 1. Potential active site residues of TcpJ,regardless of the level of conservation, are also shown in FIG. 1.

A mutational strategy was then used to determine the active siteresidues of type 4 prepilin peptidase of Vibrio cholerae (GenbankAccession No. M74708). The nucleic acid sequence of this gene isdepicted herein as SEQ ID NO:3; the amino acid sequence of thispeptidase is depicted as SEQ ID NO:4. Fifteen of the most highlyconserved active site residues were mutated to either alanine or leucinein TcpJ, the type 4 prepilin peptidase of Vibrio cholerae. The residuesselected included S18, S46, C48, C51, S65, C73, C76, S81, E88, D125,S172, D183, D189, K191, S212, and S213 (see FIG. 2). D125 and D189 werealso mutated to N and E. D183 was mutated to N. Mutations at theselected positions of tcpJ were made in pCL10, a plasmid containing tcpJand tcpA expressed from a lac promoter. These mutant constructs of thepresent invention were then introduced into Vibrio cholerae CL381, aclassical 0395 with an insertional disruption in tcpJ and an in-framedeletion of vcpD conferring a complete inability to cleave TcpA. Theresultant strains were grown under TCP-inducing conditions (LB pH 6.5,30° C.). The resulting whole cell extracts were loaded on a SDS-PAGE geland Western immunoblot was performed with antisera to TcpA. The resultsshowed that D125A and D125N and D189A and D189N were the four mutantsthat displayed a complete loss of TcpJ activity. C48A and K191A showedsome loss of activity while all other mutants did not show any loss ofcleavage activity. The chemical and structural requirements of thereactive sites were further defined by substituting E at positions D125and D189. Partial activity was maintained. Since the chemical nature ofthe D and E side groups are similar, it is believed that the side groupchemistry is responsible for the cleavage activity of the peptidase. Thechemical structure of D provides for the optimal reaction as comparedwith E.

E. coli amber suppressor strains were utilized to determine the activityof TcpJ peptidases with a number of amino acid substitutions at the 125and 189 positions. An amber (TAG) site was individually engineered intothe 125 and 189 positions of the pCL10 plasmid and the resultantconstructs were introduced into the Amber-Lys, Amber-Leu, Amber-Gln,Amber-Ser, Amber-Tyr amber suppressor strains. The strains were grown tomid-logarithmic phase and whole cell extracts were examined for TcpA andTcpJ by Western immunoblot analysis. No amino acid substitution ateither 125 or 189 exhibited any cleavage activity. The protein levels ofTcpJ were all comparable to wild-type TcpJ.

TcpJ mutant activity was then assessed in an in vitro assay modeledafter the in vitro peptidase assay devised for PilD of P. aeruginosa(Nunn, D. N. and S. Lory. 1991. Proc. Natl. Acad. Sci. USA88:3281-3285). Crude preparations of TcpA-containing membranes fromJM109 p3Z A and TcpJ-containing membranes from JM109 pCL9 were combinedwith cardiolipin and 5× assay buffer to commence cleavage of TcpA. Afterincubation of TcpA-containing membranes with approximately 0.5 μg ofTcpA prepilin and increasing amounts of TcpJ-containing membranes, 1unit of TcpJ activity was defined as the amount of TcpJ-containingmembrane required to cleave 50% of 0.5 μg of TcpA prepilin in 1 hour. Todetermine the in vitro peptidase activity of 10 of the 16 TcpJ aminoacid positions previously tested in vivo, single or multiple mutationsrepresenting each position were constructed in PCL9 and introduced intoJM109. Membrane preparations of these E. coli strains which lack TcpAprepilin were combined in the in vitro peptidase assay with 0.5 μg ofTcpA prepilin from TcpA-containing membranes. Western blot analysisdemonstrated that mutations at positions D125 and D189 were completelydefective for processing whereas all other TcpJ mutants tested had somepeptidase activity except for C48A which showed no detectable cleavageactivity. However, the C48A TcpJ protein degrades in vitro. The proteinlevels of all of the TcpJ mutants were comparable.

Since no residue was found in domain 1 that was an absolute requirementfor TcpJ cleavage activity and because several type 4 prepilin homologsdo not have domain 1 in their protein sequence, the requirement fordomain 1 of TcpJ for cleavage activity was examined. A deletion was madein domain 1 from position 35 to 81 of TcpJ using pCL11 producingpCL11Δ35-81. The deletion of 35 through 81 removed the two cysteinepairs as well as the highly conserved serines at positions 46 and 65.This deletion therefore removes the residues that had been implicated inprepilin protease activity by PilD in P. aeruginosa (Strom and Lory,1993. Ann. Rev. Microbiol. 47:565-596). pCL11Δ35-81 was introduced intoJM109 and a membrane preparation was made and tested in the in vitrocleavage assay. This assay demonstrated that the TcpJ derivativeexpressed by the deletion retained activity. Western analysis using ananti-6His antibody confirmed that TcpJΔ35-81 was expressed and stable.

To determine the peptidase activity of TcpJΔ35-81 in vivo, pCL11Δ35-81and the wild-type control pCL11 were introduced into J71K cells. Theresultant strains were grown under TCP-inducing conditions. Whole cellextracts were examined by SDS-PAGE and Western immunoblot analysis withanti-TcpA antisera. The results showed that pCL11Δ35-81 can complementJ71K similar to pCL11. TcpJΔ35-81 was able to restore TCP biogenesis toJ71K cells as seen by TEM with a negative stain of the cultures. Thesedata demonstrate that, contrary to prior art teachings (Strom, M. S. etal. 1993. Proc. Natl. Acad. Sci. USA 90:2404-2408), domain 1 is notrequired for type 4 prepilin cleavage or assembly of the resultingmature pilin into the pilus structure.

Thus, analysis of the protein sequence alignment of the type 4 prepilinpeptidase family and the mutational strategy using the TcpJ mutantconstructs coupled with the in vivo and in vitro assays confirmed thataspartic acid residues 125 and 189 make up the active site contrary toprior art teachings relating to cysteine pairs.

Based upon this identification, a standard TcpJ in vitro cleavage assaywas performed with a number of chemical protease inhibitors to examinetheir ability to block TcpJ activity. Inhibitors were selected basedupon this knowledge of the active site that were expected to effect ornot effect this peptidase activity. The effect of chemical proteaseinhibitors on the peptidase activity of TcpJ was determined byincubating the inhibitor with an amount of membrane preparation known tocontain 1 unit of activity of TcpJ for 30 minutes at room temperature.The TcpJ/inhibitor mixture was then tested for peptidase activity in thein vitro processing assay described in Example 3. Amounts of cleavedTcpA prepilin were quantitated and applied to the formula: %cleavage=(amount of cleaved TcpA in inhibition assay/amount of cleavedTcpA in no inhibitor control assay)×100. The inhibition ofEDAC/Glycinamide is a two step protocol conducted in acidic conditionsas described in Example 4. The inhibitors tested and the results areshown below in Table 1.

TABLE 1 The Effect of Chemical Inhibitors n TcpJ Cleavage In VitroSpecif- Suggested Test % Clea- Chemical icity ConcentrationConcentration vage E-64 cysteine (1.4-28 μM)^(a) 28 μM 96 proteases(1-10 μM)^(b) Calpain cysteine 45 μM 45 μM 87 inhibitor proteases I NEMcysteine 1 mM^(c) 1 mM 30 proteases Aprotinin serine (0.01-0.3 μM)^(a)0.3 μM 100 proteases 3,4-DCI serine (5-200 μM)^(a) 100 μM 89 proteases(5-100 μM)^(b) 200 μM 54 Pefabloc serine (0.4-4 mM)^(a) 2 mM 62 SCproteases 4 mM 50 Leupeptin serine and 1 μM^(a) 10 μM 85 cysteine (1-10μM)^(b) proteases PMSF serine and (0.1-1 mM)^(a) 1 mM 98 cysteine 3mM^(c) 3 mM 68 proteases Phosphor- metallo- (0.007-0.6 mM)^(a) 0.6 mM 89amidon peptidases EDTA-Na metallo- (0.5-1.3 mM)^(a) 1.3 mM 100 proteasesBestatin amino 130 μM^(a) 130 μM 81 peptidase (metallo-) Pepstatinaspartic 1 μM^(a) 1 μM 95 proteases (1-5 μM)^(b) 10 μM 92 100 μM 74EDAC/ acid 0.1 M/1 M 0.1 M/0.2 M 0 Glycin- proteases amide^(a)Boehringer Mannheim Corp. ^(b)Prolysis, a protease and proteaseinhibitor Web server, Thierry Moreau ^(c)Strom, 1993The majority of the inhibitors tested had no effect. Included in thisgroup were several inhibitors of cysteine proteases, serine proteases,metalloproteases, and one general commercially available aspartic acidprotease inhibitor, pepstatin. Others had non-specific inhibitoryeffects such as NEM, which modifies cysteines and PMSF and Pefabloc SCwhich modifies serines. These inhibitors had partial effects at thehigher test doses of the suggested concentrations. The completeinsensitivity of TcpJ activity to pepstatin rules out the possibilitythat TcpJ is a pepsin-like aspartic acid protease. A combination of EDACand glycine amide, which was selected based upon the active sitedetermination described herein and its known ability to modify asparticacid residues (Hoare, D. G. and Koshland, D. E. Jr. 1966. J. AmericanChemical Society 88(9):2057-8), inhibited TcpJ peptidase activity in thein vitro cleavage assay.

The ability to inhibit type 4 prepilin peptidase activity using peptidescorresponding to the region surrounding the cleavage site of theprepilin substrate was also examined. For these experiments, a series ofpeptides were coexpressed in bacteria in which preTcpA could beprocessed to the mature form of the TcpJ prepilin peptidase. In order toexpress only a portion of preTcpA as a peptide, an amber (Am) mutationwas created corresponding to position −1, +1, or +5 relative to thecleavage site of TcpA. Peptides were tested that were wild-type, or thatcontained mutations that would prevent cleavage by TcpJ. Their abilityto function in trans to compete for TcpJ processing activity of thecoexistent wild-type preTcpA was then assessed in a standard westernimmunoblot which detects precursor and mature forms of TcpA.

The mutations were made in a construct consisting of a 1 kb tcpJfragment followed by a 2 kb tcpA fragment on the pALTER-Ex2 backbone. Ineach of these, tcpA contained the following mutations relative to its +1processed amino acid:

Amino Acid Substitution and Position Plasmid −3 −2 −1 +1 +2 +3 +4 +5pCL1 Gln Glu Gly Met Thr Leu Leu Glu (wild-type) pCL94 Am pCL142 PropCL144 Am pCL159 Am pCL161 Pro Am pNPD-TCPA-5 Pro Leu Am pNPD-TCPA-6 ProAm pNPD-TCPA-7 Leu AmTo accomplish the analysis, these constructs were electroporated intoJM109 cells. Antibiotic resistance on the pALTER-Ex2 backbone was tochloramphenicol, with the exception of CL1 which was to tetracycline.The wild-type TcpA for which processing was assessed was expressed frompGEM-3ZA/TCP-A which confers resistance to ampicillin. Expression ofeach peptide was driven via the T7 promoter and T7 RNA polymeraseexpressed upon infection of the cells with λDE3 phage.

Once transformed, the cells in the log-phase of growth were induced byinfection with various concentrations of λDE3 phage and the whole-cellprotein extract was run out on an acrylamide gel. Western immunoblottingwith rabbit αTcpA showed both a leader-sequence processed mature TcpAband (identified relative to a 0395 TCP-A control) and a highermolecular weight unprocessed preTcpA band (identified relative to thepGEM3ZA control). In the experimental lanes, the ratio of unprocessed toprocessed forms increased with increasing expression of the peptide.These results were observed for every peptide examined, thusdemonstrating that multiple peptide derivatives corresponding to theregion surrounding the cleavage site of TcpA prepilin are capable ofinhibiting the activity of the TcpJ type 4 prepilin peptidase. By“corresponding to” it is meant that the peptide is identical to theregion surrounding the cleavage site of the target protein or substrateof the TFPP-like aspartyl protease or contains sufficient amino acidsimilarity such that the peptide mimics this region. As demonstratedherein, these peptide mimics inhibit the cleavage activity of theTFPP-like aspartyl proteases. In a preferred embodiment, these peptidemimics range in length from approximately 12 to 30 amino acids.

The combined results of the in vitro cleavage testing demonstrated thatnone of the highly conserved serines or cysteines are an active siteresidue of protease activity for TcpJ. However, D125 and D189 aredemonstrated to be protease active site residues of TcpJ, therebyclassifying TcpJ as a monomeric aspartic acid protease. The importanceof these two residue sites is further supported by the data showing thatall substitutions, except of E, at either positions 125 or 189 failed torestore any amount of protease activity indicating both aspartic acidsare required for protease activity and further defining the chemicalnature of the reaction. Therefore, TcpJ is a novel type of protease withtwo aspartic acid residues as the active site. This protease iscompletely resistant to pepstatin, a general aspartic acid proteaseinhibitor. The protein sequence analysis of the family of type 4prepilin peptidases indicates that a common protease mechanism existsamong these peptidases, regardless of the bacterial species tested.Further, it is believed that TFPP-like aspartyl proteases which utilizethe same cleavage mechanism and/or contain the same active site as type4 prepilin peptidase are present in other organisms, including humans.Such enzymes can be identified by the presence of the consensus sequenceG-X-G-D-(F or V or P or K or I or L or Y) at the C-terminal asparticacid residue.

In order to provide further evidence that the aspartic acid residuescorresponding to D125 and D189 of TcpJ comprise the peptidase activesite in all type 4 prepilin peptidases of the TFPP-like aspartylprotease subclass, mutations were also made to the putative proteaseactive site of aspartic acid residues D147 and D212 of the other type 4prepilin peptidase in V. cholerae, vcpD. Mutant D147A and D212A wereconstructed in vcpD on the plasmid pJM294. Mutant constructs D147A andD212A were examined for their ability to cleave the TcpA prepilin.Wild-type vcpD is known to be capable of at least partially cleavingthis prepilin. In these experiments, wild-type, D147A and D212A forms ofpJM294 were introduced into an E. coli JM109 strain carrying theTcpA-expressing plasmid pRTH3G7. Strains were grown to midlog phase;whole cell protein extracts were made from the cultures; and theextracts were examined by SDS-PAGE and western immunoblot analysis withanti-TcpA antiserum. Wild-type vcpD cleaved the TcpA prepilin completelyunder these conditions. In contrast, no cleavage occurred in the D147Aand D212A mutants. An analogous result was seen with the cleavage of anatural substrate of vcpD, the type 4 prepilin-like protein EpsI. Inthese experiments, wild-type, D147A and D212A forms of pJM294 wereintroduced into JM290, an E. coli JM109 strain carrying theEpsI(6)His-expressing plasmid, pJM290. Strains were grown to midlogphase under the induction of 1 mM IPTG in order to express EpsI(6)His.Whole cell protein extracts were made from the cultures and examined bySDS-PAGE and western immunoblot analysis using the Tetra-His monoclonalantibody to detect the c-terminally 6-His epitope tagged EpsI. Wild-typeVcpD was able to completely cleave EpsI from the precursor form to themature form whereas the mutants were unable to perform this cleavage.These experiments thus demonstrate that alanine substitutions at D147and D212, the aspartic acid residues in VcpD which correspond toprotease active site D125 and D189 of TcpJ, completely eliminatepeptidase activity. Thus, it is believed that these two residuescomprise the active site of all members of the type 4 prepilin peptidaseenzyme family. Accordingly, by targeting the active site identifiedherein as comprising two aspartic acid residues, one of skill canidentify compounds capable of inhibiting activity of type 4 prepilinpeptidases and other TFPP-like aspartyl proteases which utilize thissame cleavage mechanism and/or contain the same active site. Thesecompounds are expected to be useful as inhibitors of TFPP-like aspartylproteases. In one embodiment, these compounds can be used asanti-bacterial agents in the inhibition of the type 4 prepilinproteases. One example of a method for identifying such inhibitorscomprises determining the inhibitory activity of a test compound in anin vitro cleavage assay. In one embodiment of this assay, a bacterialcell membrane preparation which expresses type 4 prepilin peptidase isincubated with a test compound. The amount of cleavage activity in thepreparation in the presence of the test compound is then compared tocleavage activity in a preparation which does not contain the testcompound. Test compounds which decrease or inhibit cleavage activity areinhibitors of type 4 prepilin peptidase as well as other TFPP-likeaspartyl proteases. For example, using this method, EDAC in combinationwith glycine amide was identified as an inhibitor of type 4 prepilinpeptidase. In addition, peptides corresponding to the region surroundingthe cleavage site of the prepilin substrate were also demonstrated to beeffective inhibitors of type 4 prepilin peptidase. High throughputscreening assays can also be developed in accordance with well knownmethodologies to identify these inhibitors. Identified inhibitors arebelieved to be useful as anti-bacterial agents. These agents are alsoexpected to be useful in inhibiting the cleavage activity of otherTFPP-like aspartyl proteases in this subclass which utilize the samecleavage mechanism and/or contain the same active site.

Further, knowledge of the chemical nature of test compounds identifiedas inhibitors and the target site at which these inhibitors must act onthe enzyme to inhibit cleavage activity is useful in the identificationand/or design and development of additional inhibitory agents. Forexample, test compounds with structures known or suspected to target theaspartic acid residues of type 4 prepilin peptidase or to mimic peptidescorresponding to the region surrounding the cleavage site of theprepilin substrate can be identified. Alternatively, new test compoundswith structures designed to target aspartic acid residues of type 4prepilin peptidase or to mimic peptides corresponding to the regionsurrounding the cleavage site of the prepilin substrate can besynthesized. The ability of these test compounds to inhibit cleavageactivity of this peptidase is then determined via well known methods. Inone embodiment, inhibitory activity is determined via an in vitrocleavage assay such as that described herein. Similar approaches tothose used in the design and selection or identification of proteaseinhibitors for the treatment of HIV can also be utilized in the designand selection or identification of these new anti-bacterial agents.

Test compounds identified as inhibitors of peptidase cleavage activityare believed to be particularly useful as anti-bacterial agents.Pharmaceutical compositions comprising an active test compound and apharmaceutically acceptable vehicle can be formulated in accordance withwell known techniques. The compounds can then be administered to inhibitvirulence factor production by bacteria and to inhibit bacterialinfections in a host. By “host” it is meant to include humans. Further,it is believed that active test compounds will also be useful ininhibiting development of drug resistant strains of bacteria whenadministered in combination with a second known therapeuticallyeffective anti-bacterial agent. In this embodiment, it is preferred thatthe active test compound be administered just prior to or at the sametime as the second anti-bacterial agent. Thus, the present inventionalso relates to compositions comprising a compound which inhibits type 4prepilin peptidase activity and a second known therapeutically effectiveanti-bacterial agent.

The following non-limiting examples are provided to further illustratethe claimed invention.

EXAMPLES Example 1 Membrane Preparation

Membrane preparation protocol was adapted from the cell fractionationprotocol described by Pfau (1998. J. Bact. 180(17):4724-33). Overnightcultures were placed on ice for 20 minutes, then pelleted at 5000×g for10 minutes. The cell pellet was resuspended in 1 ml 200 mM Tris pH 8.0to which 1 ml 50 mM Tris pH 8.0, 1 M sucrose, 2 ml water, 20 μl 0.5 MEDTA, and 20 μl lysozyme (10 mg/ml) were added. The cell suspension wasallowed to incubate on ice for 30 minutes. Then, 100 μl 1 M MgSO₄ wasadded and the suspension was pelleted at 5000×g for 10 minutes. The cellpellet was resuspended in 5 ml 50 mM Tris pH 8.0, sonicated 3 times for15 seconds, and centrifuged at 5000×g for 10 minutes. The supernatantwas then centrifuged at 230,000×g for 15 minutes. The resulting pellet,which contained the total membrane fraction was resuspended in 200 μl 50mM Tris pH 8.0.

Example 2 TcpA Prepilin Quantitation

Membrane preparations were made from overnight cultures at 42° C. of theE. coli K38 that does not express TcpA and E. coli K38 p3Z-A, pGP1-2which overexpresses TcpA prepilin. P3Z-A provides tcpA under the controlof a T7 promoter, and GP1-2 provides the heat-inducible T7 RNApolymerase that drives transcription of the tcpA on p3Z-A. Then, 20 μlof the membrane preparation of K38 and K38 p3Z-A, pGP1-2 along with 3 μgof trypsin were subjected to electrophoresis on a 12.5% SDS-PAGE gelfollowed by staining with Coomassie Blue. The stained gel was driedusing the NOVEX gel drying system. The dried gel was scanned by aMolecular Dynamics Personal Densitometer SI and the densitometryanalysis was performed using ImageQuant version 1.2. The volume of the 3μg trypsin band, the TcpA prepilin band, the corresponding location ofthe TcpA prepilin band in the TcpA negative control lane (K38), and anapproximately 28 kDa band from both K38 and K39 p3Z-A, pGP1-2 weremeasured while adjusting to a single background value. The mass of theTcpA prepilin present in the K30 p3Z-A, pGP1-2 lane was determined byfirst subtracting the volume of the TcpA prepilin band from the volumeof the corresponding area in the K38 TcpA prepilin-lacking lane. Thiscontrolled for the staining intensity of minor protein bands thatco-migrate with TcpA prepilin. By comparing the volume values from the28 kDa bands from the TcpA and TcpA-negative lanes a further adjustmentwas made to the TcpA prepilin value to control for the loadingdifference between the lanes. The trypsin band volume was divided by 3μg to determine a volume/μg value, which was applied to the final TcpAprepilin band value to determine the μg TcpA/μl membrane preparation.The TcpA membrane preparation was diluted with 50 mM Tris buffer to aconcentration of 0.2 μg/μl.

Example 3 In Vitro Cleavage Assay

A membrane preparation from the E. coli strain K38 p3Z-A, pGP1-2, whichwas determined to contain 0.2 μg TcpA prepilin/μl, was the source ofprepilin in the in vitro TcpJ proteases assay instead of purifiedprepilin. A membrane preparation of the TcpJ-expressing strain JM109,pCL9, both wild-type and mutant alleles was the source of the wild-typeand mutant TcpJ protein for the in vitro cleavage reactions. The 100 μlprocessing reaction is performed by combining a 50 μl substrate fractionwith a 50 μl enzyme fraction and incubating at 37° C. for 1 hour. Thesubstrate fraction was prepared by combining 5 μl of the TcpA-containingmembrane preparation (1 μg TcpA), 10 μl 0.5% w/v cardiolipin, 20 μl of2× assay buffer (125 mM triethanolamine HCl pH 7.5, 2.5% v/vTriton-X-100), and brought up to the total volume with water. The enzymefraction was prepared by combining varying volumes of TcpJ-containingmembrane preparation and water to bring the volume to 50 μl. Thecleavage reaction was stopped by the addition of 100 μl of 5X proteinsample buffer. An increasing range of volumes of the membranepreparation containing wild-type TcpJ was added to the in vitroprocessing assay in order to determine the amount required to cleave 50%of the 1 μg TcpA included in the assay. This amount of activity wasdefined as 1 unit of TcpJ cleavage activity.

Example 4 Chemical Inhibitors

Chemical inhibitors were tested for their ability to prevent cleavage ofTcpA from the prepilin form to the mature form in the in vitro cleavageassay. Inhibitors were added at specific concentrations to the in vitroassay mixture, which contained 1 unit of TcpJ activity. Inhibitors wereadded to both enzyme and substrate fractions so that when combined theinhibitor concentration remained constant. The enzyme fraction wasallowed to incubate at room temperature for 30 minutes prior tocombination of the two fractions. The 100 μl reaction was allowed toproceed for 1 hour at 37° C. before addition of 100 μl of 2×SDS proteinsample buffer that stopped the reaction. The specific concentrations ofeach inhibitor are described in Table 1.

The EDAC/glycinamide inhibition protocol is a two step chemical reactionthat modifies carboxylic acid functional groups, such as those thatpartly make up the R-group of aspartic acid and glutamic acid. Thisprotocol is based on well known procedures set forth for the selectivemodification of carboxyl groups in proteins. TcpJ containing membranescontaining the equivalent of 6 units of TcpJ activity were incubated in80 μl with 25 mM potassium biphthalate, pH 4.0, and 100 mM EDAC(1-ethyl-3-(3-dimethlaminopropyl)carbodiimide hydrochloride) for 30minutes at room temperature. Twenty microliters of 1 M glycinamide wasadded to the mixture and another 30 minute incubation at roomtemperature was performed before the modification reaction was stoppedby the addition of 150 μl of 200 mM Tris buffer, pH 8. The mixture wasdialyzed against 50 mM Tris buffer, pH 8, for 1 hour and thencentrifuged at 230,000×g (60,000 rpm in TLA 100.3 rotor) for 15 minutes.The resulting membrane pellet, which contains the chemically modifiedTcpJ, was resuspended in 60 μl 50 mM Tris, pH 8. Twenty microliters ofthe membrane suspension was subjected to the in vitro peptidase assay todetermine the peptidase activity. Activity of the modified TcpJ wascompared to an equivalent amount of TcpJ that had been subjected to amock modification procedure which did not include EDAC or glycinamide.

1. A method of designing inhibitors of type 4 prepilin peptidase(TFPP)-like aspartyl protease enzymes comprising: (a) synthesizing acompound which targets aspartic acid residues of an enzymatic activesite of a TFPP-like aspartyl protease enzyme or mimics a regionsurrounding a cleavage site of a TFPP-like aspartyl protease substrate;and (b) determining the ability of the synthesized compound to inhibitcleavage activity of the TFPP-like aspartyl protease enzyme.
 2. Themethod of claim 1 wherein the TFPP-like aspartyl protease enzyme is atype 4 prepilin peptidase.
 3. The method of claim 2 wherein the abilityof the synthesized compound to inhibit cleavage activity is determinedvia a method comprising: (a) preparing a membrane fraction from abacterial strain expressing a type 4 prepilin peptidase; (b) contactingthe membrane fraction with the synthesized compound; and (c) determiningcleavage activity of the type 4 prepilin peptidase in the presence ofthe synthesized compound, wherein a decrease of the cleavage activity ofthe type 4 prepilin peptidase in the presence of the synthesizedcompound as compared to the activity of type 4 prepilin peptidase in theabsence of the synthesized compound is indicative of the test compoundbeing an inhibitor of type 4 prepilin peptidase.
 4. A method ofidentifying potential inhibitors of TFPP-like aspartyl protease enzymescomprising: (a) selecting a test compound having a structure known to orsuspected of targeting aspartic acid residues or mimicking a regionsurrounding a cleavage site of a TFPP-like aspartyl protease substrate;and (b) determining the ability of the identified test compound toinhibit cleavage activity of the TFPP-like aspartyl protease enzyme. 5.The method of claim 4 wherein the TFPP-like aspartyl protease enzyme isa type 4 prepilin peptidase.
 6. A mutant construct of TcpJ wherein anamino acid at position 18, 46, 48, 51, 65, 73, 76, 81, 88, 125, 172,183, 189, 191, 212, or 213 of SEQ ID NO:4 is mutated.
 7. The mutantconstruct of claim 6 wherein the amino acid is mutated to alanine orleucine.
 8. The mutant construct of claim 6 wherein the amino acid atposition 125 or 189 of SEQ ID NO: 4 is mutated to asparagine or glutamicacid.
 9. The mutant construct of claim 6 wherein the amino acid atposition 183 of SEQ ID NO:4 is mutated to asparagine.
 10. A nucleic acidsequence encoding a type 4 prepilin peptidase homologue inStaphylococcus aureus comprising SEQ ID NO:2.
 11. The nucleic acidsequence of claim 10 comprising SEQ ID NO:1.